The present invention relates to a receptor for the carboxyl-terminal cell-binding domain (CBD) of thrombospondins and, more particularly, to a novel 52 kDa protein which is a receptor for the cell-binding sequences in the CBD of thrombospondin 1 (TS1).
(Note: Literature references on the following background information, and on conventional test methods and laboratory procedures well known to the ordinary person skilled in the art, and other such state-of-the-art techniques as used herein are indicated in parentheses, and appended at the end of the application).
The thrombospondins (TS) are a family of multidomain glycoproteins (1) that influence the migration, attachment, proliferation and differentiation of a number of cell types.
The highly regulated expression of the TS isoforms during development indicates that they have important roles in developmental processes (2).
In addition, TS plays an important role in processes like wound healing (3,4), tumorigenesis (5), and angiogenesis (6-8).
The effects of TS1 and other isoforms on cells are mediated through the interaction of several domains within the complex TS structure with a number of cell surface receptors. For example, the heparin-binding domain (HBD) of TS1 binds to cell surface heparin (heparan) sulfate proteoglycans (HSPGs) (9), chondroitin sulfate proteoglycans (10), and sulfatides (11) on many different cell types.
The single RGD sequence within the type 3 or calcium-binding repeats of TS1 binds to .alpha..sub.v .beta..sub.3 integrins on some cells (12), and the Thr-Cys-Gly-containing sequence in the type 1 or properdin (malaria)-like repeats of TS1 and TS2 binds to CD36 on platelets, monocytes, endothelial cells and some tumor cells (13-15).
In addition to these sites in TS1, the carboxyl-terminal domain has been identified as a binding site for many types of normal and transformed cells (16). This domain is adjacent in the linear amino acid sequence of TS1 to the RGD sequence in the last of the type 3 repeats (17). To determine if the RGD sequence was necessary for the cell adhesion activity of the C-terminal domain, this region of TS1 was expressed in E. coli from a cDNA construct that began downstream of the RGD sequence (17). The expressed TS1 domain had substantial cell-binding activity even though the RGD sequence was excluded, indicating that this region of TS1 (referred to as the cell-binding domain or CBD) contained one or more novel cell attachment sites (17).
Using overlapping synthetic peptides representing the entire 221 amino acid residues of the CBD, two 30mer peptides with potent attachment activity toward many types of transformed and normal cells were identified (18).
In applicant's application, Ser. No. 08/029,333, filed Mar. 5, 1993, now U.S. Pat. No. 5,399,667, small VVM-containing peptides are disclosed which bind to the TS1 receptor. These peptides preferably have 5-13 amino acid residues which share the tripeptide Val-Val-Met and have the five sequences shown below as SEQ ID NOS: 1-5:
These five peptides were designated for structural purposes as 4N1, 4N1-2, 4N1-1, 7N3 and 7N3-1, respectively.
The two related sequences, 4N1-2 and 7N3-1, were disclosed to be minimal cell-binding sites. Adhesion of cells to either of these peptides is inhibited by the other, suggesting that both interact with the same receptor on cells. See also reference (19).
Various of these VVM-containing peptides, designated by the three-letter abbreviations, are shown in the Sequence Listing herein and in the accompanying Diskette as follows:
__________________________________________________________________________ Arg Phe Tyr Val Val Met Trp Lys Gln Val Thr Gln Ser [SEQ ID NO: 1] 510 Arg Phe Tyr Val Val Met Trp Lys [SEQ ID NO: 2] Arg Phe Tyr Val Val Met [SEQ ID NO: 3] 5 Phe Ile Arg Val Val Met Tyr Glu Gly Lys Lys [SEQ ID NO: 4] 510 Ile Arg Val Val Met [SEQ ID NO: 5] 5 __________________________________________________________________________